ABSTRACT To protect us from myriad diseases, adaptive immunity requires T cell recognition of protein-derived peptides of foreign, mutant, or otherwise anomalous origin expressed on the surface of aberrant cells. Central to T cell function is the cell surface ?? T cell receptor (TCR), which recognizes these various peptides bound to major histocompatibility molecules (pMHC). While a great deal is known about the static conformations of TCR molecules and their pMHC ligands as well as the resultant ligation complexes, it is still unknown precisely what happens within the TCR-pMHC complex to generate the diverse signaling outcomes which drive T cell responses. Recent experiments have highlighted the dynamic nature of TCR-pMHC ligation and signaling, with a critical input of force necessary to generate T cell responses. This implies a dynamic system, poised to signal with the input of piconewton (pN) amounts of force to generate signaling-ready TCR proteins. We propose to develop NMR methods for studies of large extracellular domains involved in TCR mechanobiology, including the TCR and its developmental precursor, the preTCR, as well as the pMHC ligands that are at the limit of NMR observation. This includes protein domains that cannot be expressed in bacterial systems and thus cannot readily be perdeuterated, a current standard labeling strategy for addressing high molecular weight protein systems. Thus, in Aim 1 we employ direct 15N-detection methods, which do not require protein perdeuteration for backbone resonance assignment. Further, we will develop new 13C-detected experiments with TROSY enhancement that yield highly resolved spectra of aromatic side chains. To augment the state of the art NMR technology above, in Aim 2 we will establish new labeling schemes to aid in deciphering the structure and dynamics of preTCR, TCR and pMHC. To tackle the resonance assignment of these large proteins we will pursue the use of ?mixed pyruvate? as a carbon source to label proteins to obtain residue specific patterns. In tandem with Aim 1 we will produce isolated 13C and 13C-19F labeled aromatic amino acids through chemoenzymatic synthesis. Since some protein components of the TCR systems we intend to study cannot be recombinantly expressed in E.Coli, we will pursue expression in eukaryotic systems, where complete deuteration is a challenge. The labeling technology developed here will be transferred to Core B. In Aim 3, we will use the NMR technologies and labeling methods, described above, to obtain information about structure and dynamics of TCR-pMHC complexes. In particular relaxation dispersion and CEST, we will leverage 19F nuclei as probe to access dynamics in the low microsecond time scale. The extracted dynamics information will be utilized with the MD Core C to observe dynamics in silico and link the dynamics and conformational states measured in NMR to those that are observed under force. Functional impact of the atomistic findings will be assessed in Projects 1 and 2 through mutational iterations. The NMR methods forged here will illuminate the proverbial blind spot in mechanistic understanding of TCR activation, already previewed via exciting preliminary hidden state data in Aim 3.